Antibodies: monoclonal and polyclonal

Background: Chlamydophila pneumoniae is an important etiological agent in respiratory system infections. The aim of study was to analyze the rate of Chlamydophila pneumoniae infection in adults and children and also to determine a correlation between the presence of this pathogen and symptoms of chronic cough. Material/Methods: The material for the study included swabs from the posterior pharyngeal wall taken on an empty stomach without cleaning the mouth. The diagnostic method was indirect immunofluorescence test (IIFT), which uses two types of antibodies: monoclonal mouse antibodies, which link specifically with the antigen that is present in the tested material and goat anti-mouse antibodies linked to fluorescein isothiocyanate, providing the colour reaction with C. pneumoniae antigen. Results: In our research, 593 patients, including 319 women, 175 men, aged from 18 to 87 years and a group of 99 children aged from 2 to 17 years with symptoms of chronic cough n=432 and other respiratory manifestations n=161 were studied. In the group of studied women with cough, 28.2% (64/227) of results were positive. In the group of men with cough, 22.3% (27/121) of results were positive. In the group of children with a cough, 28.6% (24/84) of the results were positive. Conclusions: In the examined group of children and adults with a chronic cough, the C. pneumoniae antigen was detected. The frequency of detection of C. pneumoniae antigen differed depending on the age group of both children and adults with symptoms of chronic cough.

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BROADLY NEUTRALIZING ANTI- HIV-1 ANTIBODIES DO NOT INHIBIT HIV-1 ENV-MEDIATED CELL-CELL FUSION

10.1101/2021.06.08.447628 ◽

2021 ◽

Author(s):

Nejat Düzgüneş ◽

Michael Yee ◽

Deborah Chau

Keyword(s):

Cell Fusion ◽

Neutralizing Antibodies ◽

Culture Media ◽

Genetic Material ◽

Inhibitory Effect ◽

Hippeastrum Hybrid Agglutinin ◽

Calcein Am ◽

Antibodies Monoclonal ◽

Hiv 1 ◽

Cell Cell

PG9, PG16, PGT121, and PGT145 antibodies were identified from culture media of activated memory B-cells of an infected donor and shown to neutralize many HIV-1 strains. Since HIV-1 spreads via both free virions and cell-cell fusion, we examined the effect of the antibodies on HIV-1 Env-mediated cell-cell fusion. Clone69TRevEnv cells that express Env in the absence of tetracycline were labeled with Calcein-AM Green, and incubated with CD4+ SupT1 cells labeled with CellTrace Calcein Red-Orange, with or without antibodies. Monoclonal antibodies PG9, PG16, 2G12, PGT121, and PGT145 (at up to 50 μg/mL) had little or no inhibitory effect on fusion between HIV-Env and SupT1 cells. By contrast, Hippeastrum hybrid agglutinin completely inhibited fusion. Our results indicate that transmission of the virus or viral genetic material would not be inhibited by these broadly neutralizing antibodies. Thus, antibodies generated by HIV-1 vaccines should be screened for their inhibitory effect on Env-mediated cell-cell fusion.

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A Simple and Efficient Genetic Immunization Protocol for the Production of Highly Specific Polyclonal and Monoclonal Antibodies against the Native Form of Mammalian Proteins

International Journal of Molecular Sciences ◽

10.3390/ijms21197074 ◽

2020 ◽

Vol 21 (19) ◽

pp. 7074

Author(s):

Julie Pelletier ◽

Hervé Agonsanou ◽

Fabiana Manica ◽

Elise G. Lavoie ◽

Mabrouka Salem ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Polyclonal Antibodies ◽

Simple Procedure ◽

Gene Gun ◽

Genetic Immunization ◽

Native Form ◽

Transfected Cells ◽

Native Proteins ◽

Mammalian Proteins ◽

Antibodies Monoclonal

We have generated polyclonal and monoclonal antibodies by genetic immunization over the last two decades. In this paper, we present our most successful methodology acquired over these years and present the animals in which we obtained the highest rates of success. The technique presented is convenient, easy, affordable, and generates antibodies against mammalian proteins in their native form. This protocol requires neither expensive equipment, such as a gene gun, nor sophisticated techniques such as the conjugation of gold microspheres, electroporation, or surgery to inject in lymph nodes. The protocol presented uses simply the purified plasmid expressing the protein of interest under a strong promoter, which is injected at intramuscular and intradermal sites. This technique was tested in five species. Guinea pigs were the animals of choice for the production of polyclonal antibodies. Monoclonal antibodies could be generated in mice by giving, as a last injection, a suspension of transfected cells. The antibodies detected their antigens in their native forms. They were highly specific with very low non-specific background levels, as assessed by immune-blots, immunocytochemistry, immunohistochemistry and flow cytometry. We present herein a detailed and simple procedure to successfully raise specific antibodies against native proteins.

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Monoclonal Antibodies Monoclonal antibody for Glycans as Tools for Identifying Endogenous Glycan Ligands for Human Carbohydrate-Recognition Molecules

Glycoscience: Biology and Medicine ◽

10.1007/978-4-431-54841-6_206 ◽

2014 ◽

pp. 1551-1556

Author(s):

Reiji Kannagi ◽

Naoko Kimura

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Carbohydrate Recognition ◽

Antibodies Monoclonal

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Serum Levels of Soluble Annexin II in Patients with APL

Blood ◽

10.1182/blood.v112.11.4505.4505 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4505-4505

Author(s):

Kazuo Kawasugi

Keyword(s):

Sandwich Elisa ◽

Serum Levels ◽

Annexin Ii ◽

Severe Bleeding ◽

Citrate Buffer ◽

Carbonate Buffer ◽

Tissue Plasminogen ◽

Antibodies Monoclonal ◽

Fibrinolytic Potential ◽

Normal Controls

Abstract Acute promyelocytic leukaemia (APL, M3) is associated with both a characteristic t(15;17) and severe bleeding diathesis caused by disseminated intravascular coagulation (DIC) and/or hyperfibrinolysis. It has been suggested that annexin II, a coreceptor for tissue plasminogen activator (t-PA) and plasminogen (PLG), is overexpressed on the surface of promyelocytes, leading to an increased fibrinolytic potential. However, little is known about the serum levels of annexin II in patients with APL. Therefore, we measured concentrations of annexin II in sera from patients with APL (n=10). The serum annexin II levels were determined by following methods (sandwich ELISA). The EIA plates coated for 48h at 4· with 2ug human annexin II antibodies (monoclonal) in carbonate buffer pH 9.4 respectively. The plates were then washed once with PBS and were done for non-specific blocking with 200ul/well PBS containing 1% BSA (PBSA) for 2h at room temperature (RT). After blocking, the plates were washed once with PBS. Subsequently, 100ul of samples diluted by ten times were added to the wells. After 2h incubation at RT, the plates were washed twice with PBS containing 0.05% Tween20 (PBST) and human annexin II rabbit antibodies (polyclonal) diluted 1:250 with PBSA were added to the wells. After 2h incubation at RT, the plates were washed twice with PBST and then incubated for one hour at RT with 100ul/well HRP-conjugated goat anti-rabbit Ig diluted 1:250 in PBSA. The plates were washed twice with PBST and reaction was developed by adding 100ul/well of substrate solution (0.04% O-phenylenediamine in phosphate/citrate buffer pH 5, with 0.04% H2O2). The reaction was stopped after 10 min by adding 50ul/well 2.5M H2SO4, the absorbance was read at 492nm using a spectrophotometer In the patients with APL, we found significant elevations in serum annexin II levels as compared with normal controls. In other words, serum levels of annexin II were 0.109 (mean OD values, n=10) from patients with APL. On the other hand, concentrations of annexin II in sera from normal controls were 0.092 (mean OD values, n=10). There was correlation between the FDP and annexin II levels in the APL patients with DIC The present work demonstrates significant elevations in serum annexin II levels in patients with APL. Therefore, we suggest that elevation of annexin II may be associated with the development of an increased fibrinolytic potential.

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Q-fever presenting as an autoimmune disease: case report and review

Open Medicine ◽

10.2478/s11536-011-0097-y ◽

2011 ◽

Vol 6 (6) ◽

pp. 727-731 ◽

Cited By ~ 1

Author(s):

Maria Gamaletsou ◽

Achilleas Gikas ◽

Nikolaos Sipsas

Keyword(s):

Autoimmune Disease ◽

Monoclonal Gammopathy ◽

Erythema Nodosum ◽

Q Fever ◽

Antineutrophil Cytoplasmic Antibodies ◽

Laboratory Findings ◽

Chronic Q Fever ◽

Disease Case ◽

Antibodies Monoclonal ◽

Autoimmune Phenomena

AbstractQ fever is a worldwide zoonosis caused by the intracellular bacterium Coxiella burnetti. Autoimmune phenomena associated with the disease may obscure the clinical picture, and in many reports mislead physicians to an initial diagnosis of an autoimmune disease. We present a case of chronic Q-fever, complicated by myocarditis/pericarditis, where patient’s initial signs, symptoms and laboratory findings (i.e., protracted fever, oligoarthritis, erythema nodosum, positive antineutrophil cytoplasmic antibodies, monoclonal gammopathy) seemed to suggest an autoimmune disease. We also review the literature for autoimmune phenomena associated with Q-fever.

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Origins and patterning of avian outflow tract endocardium

Development ◽

10.1242/dev.111.4.867 ◽

1991 ◽

Vol 111 (4) ◽

pp. 867-876 ◽

Cited By ~ 10

Author(s):

D.M. Noden

Keyword(s):

Outflow Tract ◽

Avian Embryo ◽

Endocardial Cushions ◽

Head Mesoderm ◽

Developing Heart ◽

History Of ◽

Otic Placode ◽

Species Specific ◽

Antibodies Monoclonal ◽

Lateral Mesoderm

Outflow tract endocardium links the atrioventricular lining, which develops from cardiogenic plate mesoderm, with aortic arches, whose lining forms collectively from splanchnopleuric endothelial channels, local endothelial vesicles, and invasive angioblasts. At two discrete sites, outflow tract endocardial cells participate in morphogenetic events not within the repertoire of neighboring endocardium: they form mesenchymal precursors of endocardial cushions. The objectives of this research were to document the history of outflow tract endocardium in the avian embryo immediately prior to development of the heart, and to ascertain which, if any, aspects of this history are necessary to acquire cushion-forming potential. Paraxial and lateral mesodermal tissues from between somitomere 3 (midbrain level) and somite 5 were grafted from quail into chick embryos at 3–10 somite stages and, after 2–5 days incubation, survivors were fixed and sectioned. Tissues were stained with the Feulgen reaction to visualize the quail nuclear marker or with antibodies (monoclonal QH1 or polyclonals) that recognize quail but not chick cells. Many quail endothelial cells lose the characteristic nuclear heterochromatin marker, but they retain the species-specific epitope recognized by these antibodies. Precursors of outflow tract but not atrioventricular endocardium are present in cephalic paraxial and lateral mesoderm, with their greatest concentration at the level of the otic placode. Furthermore, the ventral movement of individual angiogenic cells is a normal antecedent to outflow tract formation. Cardiac myocytes were never derived from grafted head mesoderm. Thus, unlike the atrioventricular regions of the heart, outflow tract endocardial and myocardial precursors do not share a congruent embryonic history. The results of heterotopic transplantation, in which trunk paraxial or lateral mesoderm was grafted into the head, were identical, including the formation of cushion mesenchyme. This means that cushion positioning and inductive influences must operate locally within the developing heart tubes.

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Biological Properties and Cell Tropism of Chp2, a Bacteriophage of the Obligate Intracellular Bacterium Chlamydophila abortus

Journal of Bacteriology ◽

10.1128/jb.184.10.2748-2754.2002 ◽

2002 ◽

Vol 184 (10) ◽

pp. 2748-2754 ◽

Cited By ~ 19

Author(s):

J. S. Everson ◽

S. A. Garner ◽

B. Fane ◽

B.-L. Liu ◽

P. R. Lambden ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Biological Properties ◽

Enzyme Linked Immunosorbent Assay ◽

Cell Tropism ◽

Obligate Intracellular ◽

Chlamydophila Abortus ◽

Phylogenetic Studies ◽

Chlamydial Species ◽

Development Cycle ◽

Antibodies Monoclonal

ABSTRACT A number of bacteriophages belonging to the Microviridae have been described infecting chlamydiae. Phylogenetic studies divide the Chlamydiaceae into two distinct genera, Chlamydia and Chlamydophila, containing three and six different species, respectively. In this work we investigated the biological properties and host range of the recently described bacteriophage Chp2 that was originally discovered in Chlamydophila abortus. The obligate intracellular development cycle of chlamydiae has precluded the development of quantitative approaches to assay bacteriophage infectivity. Thus, we prepared hybridomas secreting monoclonal antibodies (monoclonal antibodies 40 and 55) that were specific for Chp2. We demonstrated that Chp2 binds both C. abortus elementary bodies and reticulate bodies in an enzyme-linked immunosorbent assay. Monoclonal antibodies 40 and 55 also detected bacteriophage Chp2 antigens in chlamydia-infected eukaryotic cells. We used these monoclonal antibodies to monitor the ability of Chp2 to infect all nine species of chlamydiae. Chp2 does not infect members of the genus Chlamydia (C. trachomatis, C. suis, or C. muridarum). Chp2 can infect C. abortus, C. felis, and C. pecorum but is unable to infect other members of this genus, including C. caviae and C. pneumoniae, despite the fact that these chlamydial species support the replication of very closely related bacteriophages.

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The End of Cytotoxics?

European Oncology & Haematology ◽

10.17925/eoh.2011.07.04.228 ◽

2011 ◽

Vol 07 (04) ◽

pp. 228

Author(s):

Nalân Utku ◽

Keyword(s):

Bone Marrow ◽

Cancer Research ◽

Monoclonal Antibodies ◽

Immune System ◽

Side Effects ◽

Cancer Cells ◽

Cancer Chemotherapy ◽

Targeted Therapeutics ◽

Dividing Cells ◽

Antibodies Monoclonal

Cytotoxic drugs were the first form of cancer chemotherapy to be established, but they can have quite severe side effects. Cytotoxics attack all rapidly dividing cells, including healthy cells and cancer cells. They can therefore cause side effects such as loss of hair, damage to the skin and mucosa, and damage to bone marrow, affecting the immune system. For this reason, the focus of some cancer research has moved from cytotoxics to targeted therapeutics, including monoclonal antibodies. Monoclonal antibodies can be very effective in patients who express the appropriate target; however, not all patients do, and some develop resistance. Monoclonal antibodies, like other biologicals, are also very costly. All is not lost for cytotoxics. Because they have been around for so long, there is an abundance of data on their modes of action. Based on these data, researchers have developed newer cytotoxics that are more effective and have fewer side effects, and approaches that deliver cytotoxics more safely or target them to tumours.

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VPAC1 Receptor-Mediated Regulation of Megakaryopoiesis.

Blood ◽

10.1182/blood.v104.11.735.735 ◽

2004 ◽

Vol 104 (11) ◽

pp. 735-735

Author(s):

Kathleen Freson ◽

Chantal Thys ◽

Christine Wittevrongel ◽

Rita De Vos ◽

Jos Vermylen ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Examination ◽

T Test ◽

Bleeding Complications ◽

Bleeding Tendency ◽

Regulatory Pathways ◽

Normal Platelet ◽

Vpac1 Receptor ◽

Life Threatening ◽

Antibodies Monoclonal

Abstract Identification of the regulatory pathways that direct megakaryopoiesis and platelet production is essential for the development of novel strategies to treat life threatening bleeding complications in bone marrow suppressed patients. We demonstrated that megakaryocytes and platelets express the Gs-coupled VPAC1 receptor, for which both PACAP and VIP are specific agonists. We have further identified a bleeding tendency and found three copies of the PACAP gene in two related patients with severe mental retardation, responsible for elevated PACAP plasma levels and associated increased platelet cAMP concentrations, resulting in strongly reduced platelet aggregation (JCI, 2004, 113, 905). In this study, we have further demonstrated a fundamental role for the VPAC1 signalling pathway during megakaryocyte maturation and platelet formation. Patients with PACAP overexpression have mild thrombocytopenia, a normal platelet survival and relatively small platelets with an MPV of 8.2 fL (normal MPV 9-13 fL). FACS analysis of the patients’ platelets showed reduced expression of GPIX and GPIIIa. Electron microscopy of bone marrow of patients and of mice, specifically overexpressing PACAP in megakaryocytes revealed the presence of early megakaryocyte progenitors but almost not of mature megakaryocytes. Immature megakaryoblasts seemed to have reduced levels of rough endoplasmic reticulum cisternae and free ribosomes. To further study the modulating role of VPAC1 in thrombopoiesis, control mice were therefore subcutaneously injected with neutralizing polyclonal or monoclonal anti-PACAP, anti-VIP or anti-VPAC1 antibodies. Injection of these antibodies in all cases led to increased platelet counts, compared to control antibodies (monoclonal anti-PACAP antibody: 1194 ± 237 x 109 plt/L; control antibody: 722 ± 178 x 109 plt/L; p=0.01, unpaired t-test at day 7 after injection). This strategy was also capable of reducing the drop in platelet count in busulfan treated mice (polyclonal anti-PACAP antibody: 561 ± 121 x 109 plt/L; control antibody: 349 ± 65 x 109 plt/L; p=0.033, unpaired t-test at day 29 or day 18 after respectively antibody and busulfan injection). In addition, bone marrow examination of mice injected with monoclonal anti-PACAP or anti-VPAC1 antibodies revealed an increase in megakaryocyte numbers and showed a marked expansion and mobilization of megakaryocyte progenitor cells. Mice injected with a monoclonal anti-VPAC1 antibody showed an increase of about 50% in bone marrow CFU-MK. In conclusion, we provide evidence that the VPAC1 pathway modulates normal megakaryopoiesis. Further studies are needed to evaluate whether this pathway can be safely manipulated in man in the treatment of thrombocytopenia.

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